Oxidovanadium (IV) and iron (III) complexes with O2N2 donor linkage as plausible antidiabetic candidates: Synthesis, structural characterizations, glucose uptake and model biological media studies

Article abstract of DOI:10.1002/aoc.5327

With the upsurging cases of type II diabetic patients, the demand for safe and effective oral antidiabetic drugs is also increasing. Coordination complexes have proven their mettle as efficient oral drug candidates, which thereby motivated us in this work

Full text of DOI:10.1002/aoc.5327

Received: 17 August 2019
DOI: 10.1002/aoc.5327
Revised: 1 October 2019
Accepted: 2 October 2019
F U L L P A P E R
Oxidovanadium (IV) and iron (III) complexes with O2N2
donor linkage as plausible antidiabetic candidates:
Synthesis, structural characterizations, glucose uptake and
model biological media studies
Manasa Kongot1 | Dinesh S. Reddy1 | Vishal Singh2 | Rajan Patel3
Nitin Kumar Singhal2 | Amit Kumar1
|
1Centre for Nano and Material Sciences,
With the upsurging cases of type II diabetic patients, the demand for safe and
effective oral antidiabetic drugs is also increasing. Coordination complexes
have proven their mettle as efficient oral drug candidates, which thereby moti-
vated us in this work to design new transition metal complexes as plausible
candidates for the treatment of diabetes. A reduced salen ligand, {H2(hpdbal)2-
an} (1) derived vanadium (IV) and iron (III) complexes, namely,
[VIVO{(hpdbal)2-an}] (2) and [{FeIII (OH2)((hpdbal)2-an)}2 μ2-SO4] (3) were
synthesized in this study. The newly obtained ligand and complexes were char-
acterized using usual analytical and spectroscopic techniques. The potential of
these compounds in inducing increased glucose uptake by diabetic cells were
studied by using insulin resistant HepG2 cells as model diabetic cells and 2-
NBDG molecule as a D-glucose analogue and fluorescent tracker. The cells
added with the vanadium (IV) complex 3 induced significant NBDG uptake of
95.4% which was higher than that induced by metformin, the standard anti-
diabetic drug. To elucidate the behavior of the complexes in biological media,
model solution studies were conducted with a wide range of pH conditions
and protein bovine serum albumin (BSA). The complexes demonstrated effec-
tive binding with BSA which was concluded through spectroscopic titration
studies and were also found to be sufficiently stable over physiological pH con-
ditions. The study can thus prove to be beneficial in the quest for new anti-
diabetic drugs.
Jain University, Jain Global Campus,
Bengaluru, 562112, Karnataka India
2National Agri Food Biotechnology
Institute, Mohali, 140306, India
3Biophysical Chemistry Laboratory,
Centre for Interdisciplinary Research in
Basic Sciences, Jamia Millia Islamia (A
Central University), New Delhi, 110025,
India
Correspondence
Amit Kumar, Centre for Nano and
Material Sciences, Jain University, Jain
Global Campus, Bengaluru, 562112,
Karnataka, India.
Email: amit.kumar@jainuniversity.ac.in
Funding information
Nano Mission Council, Department of
Science and Technology, Grant/Award
Number: SR/NM/NS-20/2014; Science and
Engineering Research Board, Grant/
Award Number: SB/FT/CS-100/2013;
Nanomission project, Grant/Award
Number: SR/NM/NS-20/2014;
Department of Science and Technology
(DST), Grant/Award Number: BT/
PR10353/PFN/20/889/2013; Science and
Engineering Research Board (SERB),
India, Grant/Award Number: 100/2013
K E Y W O R D S
antidiabetic, BSA interaction, O2N2 donor ligand, oxovanadium (IV) complex, sulphate bridged
iron (III) complex
1 | INTRODUCTION
condition which leads to organ damage, dysfunction and
failure.1a In 2014, 422 million adults all over the world
Diabetes mellitus or more commonly known as ‘sugar’ is
a chronic metabolic disorder characterized by high levels
of blood glucose. This is a serious and globally prevailing
were living with diabetes. The global prevalence has dou-
bled from 4.7% in 1980 to 8.5% in 2014. According to the
recent statistics reported by the International Diabetes
Appl Organometal Chem. 2019;e5327.
wileyonlinelibrary.com/journal/aoc
© 2019 John Wiley & Sons, Ltd.
1 of 12
https://doi.org/10.1002/aoc.5327

2 of 12
KONGOT ET AL.
Federation for the year 2017, the number has raised to
425 million people and it is expected that by 2045, this
disease will take a toll on 629 million people.1b Type 2
diabetes also termed previously as adult-onset or non-
insulin dependent diabetes is the most serious and preva-
iling type and in the recent days, majority of people suffer
with this type of diabetes owing to depleting standards of
lifestyle. Many successful approaches for the treatment of
elevated blood glucose levels have been accomplished
due to extensive research and development studies. Sev-
eral classes of oral drugs[2] such as sulfonylureas,
biguanides, glucosidase inhibitors, DPP-4 inhibitors and
SGLT2 inhibitors have been established in the past few
decades which have been able to help the body to control
blood glucose levels. Although these classes of drugs have
been successful, they pose many adverse side effects such
as abdominal and gastrointestinal discomforts, diarrhoea,
drug intolerance, hypoglycaemia, weight gain, hepatotox-
icity, bone density reduction and fracture risks, nausea,
cardiovascular disease risks and genital infections.[3–7]-
Medicinal inorganic chemistry is one of the hopes for
overcoming the glitches posed by existing organic
drugs,[8] speaking of which, trace metals such as vana-
dium and iron have huge scope and have been studied
for their biological and therapeutic activities. Vanadium
constitutes 0.015% of earth's crust and is well distributed
in nature.[9,10] Several ascidian sea squirts and amanita
mushrooms are known to accumulate vanadium in the
form of vanadate (vanadium(V)oxoanion) and amavadin
(non-oxovanadium (IV) complex) respectively.[11,12]
Vanadium containing enzymes such as vanadium
bromoperoxidases occur in many marine algae, fungi,
eukaryotes and they function as a catalyst for the forma-
tion of most of the naturally available brominated com-
pounds.[13] Citing these biological significances of
vanadium, researchers have developed many nature-
mimicking and synthetic vanadium compounds for
exploration of their biological activities mainly towards
therapeutic usages. More than a century ago, a vanadium
compound, sodium vanadate was used as a medicinal
agent for the treatment of elevated blood glucose levels in
body.[14] After nearly a century leap, in 1985, Heyliger
and his group's first report[15] on the blood glucose lower-
ing effect of sodium vanadate in streptozotocin (STZ)-
induced diabetic rats generated high scientific inquisitive-
ness amongst researchers about the use of vanadium
compounds in treatment of diabetes. This curiosity led to
the development of bis (maltolato)oxovanadium (IV)
(BMOV) and its analogue bis (ethylmaltolato)
oxovanadium (IV) (BEOV) which have proved their suit-
ability as insulin enhancing agents with improved bio-
availability and have successfully undergone pre-clinical
testing for efficacy and safety.[16,17] The latter has
effectively completed phase I clinical trials and is under
study in the phase II trials for treatment of type II diabe-
tes patients.[18]
A
vanadium picolinato complex,
[VO2dipic−] is another potent antidiabetic agent which
has helped in curing cats affected with diabetes.[19] The
pyridine oxothiolato based complex, [VO (opt)2] and
acetylacetone based complex, [VO (acac)2] have exhibited
robust insulin mimetic activity by normalizing blood glu-
cose levels in STZ-rats upon oral administration.[20,21]
Numerous oxidovanadium (IV/V) complexes derived
from salen and salan type of ligands have exhibited excel-
lent in vitro and in vivo insulin enhancing activity by low-
ering blood glucose levels in STZ induced-rats. VIVO
(salen) can reverse hyperglycemia to normal glucose
levels in alloxan-induced diabetic rats.[22] Another trace
metal iron is the fourth most common element constitut-
ing about 5% of the Earth's crust.[23] Iron is required for
smooth and normal functioning of many biological pro-
cesses.[24] In humans, haemoglobin, cyctochrome and
catalase are three important biomolecules consisting of
iron in them.[25] These iron containing proteins are
responsible for transport, storage and use of oxygen, elec-
tron transfer and biocatalysis reactions. One of the very
first reports of therapeutic uses of iron was that of ferro-
cenium salts which exhibited exceptional anticancer
activities.[26,27] Following this, many iron complexes have
been reported for their exceptional anticancer and
antibacterial activities.[28,29] Especially, salen tethered
iron (III) complexes have shown detrimental effects on
cancer cells whose activities were higher than cisplatin
drug.[30,31] However, there have been no significant
reports on the antidiabetic potential of iron complexes.
To address the impending type II diabetes disease
burden, in the present study, new oxidovanadium (IV)
and iron (III) complexes affixed with a ‘salan’ type of
ligand have been prepared (Scheme 1). The moieties were
preliminarily screened for their antidiabetic activity
through glucose uptake studies on model diabetic cells
(insulin resistant HepG2 cells). The response of these
compounds in biological media was stipulated by study-
ing their behavior in varied pH conditions and protein
(BSA) solution.
2 | EXPERIMENTAL METHODS
2.1 | Materials and
instrumentation used
For all the experiments, solvents and chemicals used
were of synthesis grade and were used as received.
Vanadyl acetylacetonate, [VO (acac)2] was prepared by
following
a
very well-known procedure[32] using

ANTIDIABETIC AND BIOLOGICAL MEDIA STUDY OF V(IV) AND FE(III) COMPLEXES
3 of 12
SCHEME 1 Representation of the
complexes obtained in this study and the
biological activity studies performed for them
vanadium pentoxide (V2O5) and acetyl acetone. Ferrous
sulphate heptahydrate (purity >98%) was obtained from
S D Fine-Chem Ltd., India. Bovine serum albumin (BSA,
purity >99%, fraction V, fatty acid free, lyophilized pow-
der) was purchased from Sisco Research Laboratories
Pvt. Ltd., India. 2-hydroxy-5-(phenyldiazenyl)benzalde-
hyde (Hhpdbal) was prepared by following the method
used in our previously published work.[33] The synthetic
protocols followed for obtaining the reduced imine ligand
system and their respective vanadium and iron com-
plexes are outlined in detail in Section 3.4.
Elemental analysis was done using a CHN elemental
analyzer, Euro Vector E-3000 and energy dispersive X-ray
analyzer, JEOL-JSM7100F. Infrared spectra were mea-
sured using a Bruker Alpha Single reflection ATR setup
equipped with ZnSe crystal. Electronic spectra were mea-
sured using a UV–Vis spectrophotometer, Shimadzu UV-
1800. NMR spectra, 1H-NMR and 13C-NMR were
recorded for the ligand using an Agilent 400MR DD2 FT-
NMR Spectrometer and tetramethylsilane as internal
standard. Electron paramagnetic resonance spectra, X-
band, were obtained from a ESR Spectrometer, JEOL
JES-FA200 at 77.2 K. Thermal analysis of the complexes
was performed on a TG/DTA analyzer, Perkin Elmer
STA 6000 up to 700 ꢀC.
confluency and then treated with insulin resistance
inducing mixture of tumor necrosis factor (TNF-α), fruc-
tose and palmitic acid. These cells were then added with
different concentrations of the test complexes 2 and 3.
The cells added with well-known antidiabetic drug met-
formin and ligand 1 were taken as standard and reference
respectively for the experiments. HepG2 cells which were
not added with any insulin resistance media and study
compounds were taken as control for the experiments.
The cells which were induced with insulin resistivity
were taken as the test for the experiments. All the cells
were incubated for 24 hr and thereafter the wells were
washed with buffer PBS (1X). In succession, the fluores-
cent analogue of D-glucose, 2-NBDG (40 μM) and insulin
(0.1 μM) were added and incubated for an hour. The
wells were then carefully washed with PBS and lysis
buffer was added. The fluorescence given out from each
well were carefully measured using a fluorescence
spectrophotometer.
2.3 | Protocol for model biological media
studies
Varied pH experiments were conducted on the solution
of the synthesized complexes and for this, pH meter cali-
brated with standard buffers and UV–Vis spectrometer
were used. The mode of interaction of the compounds
with a serum protein, bovine serum albumin (BSA) was
studied using steady-state, synchronous, three-dimen-
sional fluorescence spectroscopy techniques, temperature
probed UV–Visible spectroscopy and infrared spectros-
copy techniques. The instruments used were a Varian
Cary Eclipse spectrofluorometer, Analytik Jena Specord-
250 spectrophotometer and Specac golden gate ATR
instrument. The stock solutions of BSA (5 μM) and
2.2 | Antidiabetic/glucose-uptake studies
The glucose uptake activity induced by the complexes
was evaluated with the help of 2-NBDG assay. This assay
was performed by following previously established proto-
col.[34] The model cells used for this study were human
liver carcinoma cells, HepG2 which were induced with
insulin resistivity to mimic diabetic environment. For
this, the cells were grown in a 96-well plate up to 90%

4 of 12
KONGOT ET AL.
complexes 2, 3 (1 mM each) were prepared in sodium
phosphate buffer of pH 7.4 and DMSO respectively.
(N=N), 1286 (C-N), 3053 (aromatic C-H), 1231 (C-O); 1H-
NMR (400 MHz, CDCl3): δ 13.75 (s, 2H, aromatic O-H),
8.50 (s, 2H, N=C-H), 7.98–7.05 (m, 16H, aromatic H),
4.03 (s, 4H, -CH2-CH2); 13C-NMR (400 MHz, CDCl3): δ
59.37 (2C), 118.04 (2C), 122.51 (6C), 126.95 (2C), 127.35
(2C), 128.99 (4C), 130.39 (2C), 145.33 (2C), 152.64 (2C),
164.23 (2C), 166.37 (2C).
{H2(hpdbal)2-an} (1): Yield: 1.87 g, 78%; m.p:
238 ꢀC; Anal. calcd. For. C28H28N6O2 (MW:
480.56 g mol−1): C, 69.98; H, 5.87; N, 17.49. Found: C,
69.96; H, 5.84; N, 17.52; Selected ATR-IR data (ν/cm−1):
1598, 1444 (N=N), 1285 (C-N), 3023 (aromatic C-H), 2757
(aliphatic C-H), 1158 (C-O), 3376 (N-H); 1H-NMR
(400 MHz, DMSO-d6): δ 8.04 (s, 2H, aromatic O-H), 7.85–
7.19 (m, 16H, aromatic H), 4.20 (s, 4H, Ar-CH2-N), 3.37
(s, 4H,-CH2-N); 13C-NMR (400 MHz, DMSO-d6): δ 43.58
(2C), 45.79 (2C), 116.49 (2C), 119.68 (2C), 122.55 (4C),
125.84 (2C), 126.94 (2C), 129.85 (4C), 131.16 (2C), 145.29
(2C), 152.49 (2C), 160.00 (2C).
2.4 | Synthesis
2.4.1 | Synthesis of O2N2 donor ligand,
2,20-((ethane-1,2-diylbis (azanediyl))bis
(methylene)) bis (4-(phenyldiazenyl)
phenol) {H2(hpdbal)2-an} (1)
To a solution of 2-hydroxy-5-(phenyldiazenyl)benzalde-
hyde (2.26 g, 10 mM) in 20 ml of dichloromethane, added
ethylene diamine (0.33 ml/0.30 g, 5 mM) slowly. This
reaction mixture was kept for stirring at room tempera-
ture for 4 hr (Scheme 2). Golden yellow colored precipi-
tate was obtained which was isolated after filtration
under vacuum. Subsequently, it was washed with dic-
hloromethane, diethyl ether and left for drying in a hot
air oven at 60 ꢀC to obtain the product A.
A solution of the imine compound, {H2(hpdbal)2-en}
(A) (2.38 g, 5 mM) in 15 mL of methanol and dic-
hloromethane mixture was maintained at 0 C. To this
2.4.2 | Synthesis of vanadium (IV)
complex, [VIVO{(hpdbal)2-an}] (2)
ꢀ
solution, a methanolic solution of sodium borohydride
(0.45 g, 12 mM) containing few drops of conc. KOH
solution was slowly added with stirring (Scheme 2). The
pH was set to 6 by adding required amount of HCl and
the reaction mixture was stirred for about 6–7 hr. TLC
monitoring confirmed gradual decrease in the imine
content and gradual increase in the amine content fol-
lowing reduction. As soon as the TLC spot for the imine
compound disappeared, the solvent in the reaction mix-
ture was evaporated to dryness and about 10 ml of water
was added to the residue followed by pH maintenance of
4–5. The obtained yellow product was vacuum filtered,
washed with distilled water, diethyl ether and left for
drying in a hot air oven at 50 ꢀC to obtain the new
ligand, 1.
Vanadyl acetylacetonate, [VIVO (acac)2] (0.56 g, 2.1 mM)
was taken in 20 ml of methanol and to this was added a
solution of ligand, {H2(hpdbal)2-an} (1) (0.96 g, 2.0 mM)
dissolved in 20 ml of methanol. The reaction mixture was
refluxed for 3 hr with stirring (Scheme 3). The initial light
brown colored solution turned into dark brown and this
solution was reduced to half of its volume after reflux.
The solution was then filtered to get a clear dark brown
solution. This filtrate upon room temperature evapora-
tion gave dark brown colored precipitate of 2 which was
separated, washed with diethyl ether and driedꢀin anhy-
drous conditions. Yield: 0.83 g, 76%; m.p: 278 C; Anal.
calcd. For. C28H26N6O3V(MW: 545.49 g mol−1): C, 61.65;
H, 4.80; N, 15.41. Found: C, 61.62; H, 4.82; N, 15.38;
Selected ATR-IR data (ν/cm−1): 3418 (N-H), 1470, 1579
(N=N), 1108 (C-O), 1358 (C–N), 3066–3037 (aromatic C-
H), 2934–2872 (aliphatic C–H), 982 (V=O); UV–Vis
(λmax/nm): 347, 448, 612.
{H2(hpdbal)2-en} (A): Yield: 3.90 g, 82%; Anal.
calcd. For. C28H24N6O2 (MW: 476.53 g mol−1): C, 70.57;
H, 5.08; N, 17.64. Found: C, 70.43; H, 5.01; N, 17.59;
Selected ATR-IR data (ν/cm−1): 1630 (C=N), 1592, 1484
SCHEME 2 Synthetic route to obtain
the ligand used in the present study,
{H2(hpdbal)2-an} (1)

ANTIDIABETIC AND BIOLOGICAL MEDIA STUDY OF V(IV) AND FE(III) COMPLEXES
5 of 12
SCHEME 3 Synthetic route to obtain [VIVO{(hpdbal)2-an}] (2)
2.4.3 | Synthesis of iron (III) complex,
[{FeIII (OH2)((hpdbal)2-an)}2 μ2-SO4] (3)
vanadium and iron was confirmed in compounds 2 and 3
respectively (Figure 1).
The important functional groups present in the com-
pounds were confirmed by the characteristic IR spectra
(Figure 2). The ligand H2(hpdbal)2-an (1) exhibited a typ-
ically broad phenolic O-H stretching vibrational peak
and secondary amine N-H stretching at 3307 and
3376 cm−1 respectively. The corresponding vanadium
complex, [VIVO{(hpdbal)2-an}] (2) did not show any peak
The ligand, {H2(hpdbal)2-an} (1) (0.96 g, 2.0 mM) was dis-
solved in 20 ml methanol and to this, methanolic ferrous
sulphate heptahydrate (0.58 g, 2.1 mM) solution was
added. The reaction mixture immediately turned to dark
brown color and this mixture was refluxed for 1 hr to
obtain a dark brown colred precipitate of complex 3
(Scheme 4). The hot solution was filtered to separate out
the residue which was then washed with methanol and
diethyl ether. The product obtained was then dried inꢀan
ꢀ
hot air oven at 70 C. Yield: 1.86 g, 78%; m.p: 232 C;
Anal.
calcd.
For.
C56H56N12O10SFe2
(MW:
1200.87 g mol−1): C, 56.01; H, 4.70; N, 14.00. Found: C,
55.94; H, 4.71; N, 14.09; Selected ATR-IR data (ν/cm−1):
1068 (S=O), 1391 (SO4), 1480, 1556 (N=N), 1103 (C-O),
1305 (C–N), 3003 (aromatic C–H), 2883–2996 (aliphatic
C-H), 3332 (O-H), 1639, 3242 (N-H); UV–Vis (λmax/nm):
347, 461, 624.
3 | RESULTS AND DISCUSSION
3.1 | Characterization
The elemental analysis of the newly obtained ligand and
complexes were done with the help of CHN and EDS
analysis. The data obtained for the basic elemental com-
position of the compounds were found to be in agree-
ment with the calculated values. The presence of metals,
FIGURE 1 Representative EDS analysis which confirms the
elemental composition of the ligand (1) and complexes (2 and 3)
SCHEME 4 Synthetic route to obtain [{FeIII (OH2)((hpdbal)2-an)}2 μ2-SO4] (3)

6 of 12
KONGOT ET AL.
The X-band EPR spectra of the vanadium (IV) com-
plex 2 and iron (III) complex 3 were recorded in DMSO
solution (5 × 10−4 M) at 77 K owing to the paramagnetic
nature of the V (IV) and Fe (III) species. Since the
nuclear spin of V+4 is 7/2, a typical EPR spectrum of a
vanadium (IV) complex should consist of 8 hyperfine
splitting lines and this was evident from the EPR spec-
trum obtained for the complex 2 (Figure 3). This spec-
trum was simulated manually to calculate the spin-
Hamiltonian parameters which are given in table 1. The
compound exhibited an axial spectrum with
gx = gy = g┴ > gz = g . The hyperfine splitting constant
k
(Az = A ) value obtained from the spectrum was corre-
k
lated with the number and type of ligand environment
equatorially present around the vanadyl ion using the
“additivity” rule proposed by Holyk and Chasteen.[35]
The obtained A value of 157.7 x 10−4 cm−1 matched
k
very well with the theoretically obtained value for A
k
FIGURE 2 Vibrational spectra representing the stretching
and bending modes of functional groups present in the ligand and
complexes
using the additivity rule. The value corresponds to two
amine, Namine and two OArO−, OArO− equatorially placed
donor sets which is in accordance with the expected
N
structure of the vanadyl complex 2. All the spin parame-
1
ters obtained are characteristic of dxy configuration of
for O-H stretching which confirms the participation of
phenolate O in bonding to vanadium center by losing its
proton. In addition, a signal for the vanadium-oxo bond
stretching was observed at 982 cm−1. No significant
changes were observed for other vibrational bands and
all peaks pertaining to other parts of the ligand appeared
within the anticipated range. In the IR spectra of iron
complex, [{FeIII (OH2)((hpdbal)2-an)}2 μ-O2SO2] (3), shifts
were observed for C-O and N-H stretching corresponding
to the phenolato oxygen and amine nitrogen which coor-
dinates to the metal center. O-H stretching bands were
observed corresponding to the coordinated water mole-
cules. Notably, a strong band for S=O stretching at
1068 cm−1 and peak particular to the SO4 group at
1391 cm−1 were observed. This accounts for the sulphate
bridged (μ2-SO4) coordination with two iron metal cen-
ters. All other stretching and bending vibrations of the
complex stayed intact which confirms the complexation
by the ligand.
the VO2+ ion in an axially compressed fashion[36] and the
oxovanadium (IV) complex exists in a slightly distorted
square pyramidal geometry[37] around the phenolato and
amine donor sets. The Fe (III) species with a nuclear spin
of I = 1/2 gave an anisotropic and complex splitting pat-
tern with the g value centered at 2.03 which suggested an
octahedral geometry and low spin Fe (III) metal
center.[38]
Distinct weight loss steps could not be identified in
the TGA analysis profile of the vanadium complex 2. The
thermal degradation steps involved firstly the loss of
The excitation properties of the compounds were
studied by their electronic spectra recorded in DMSO (SI
Figure 1). The UV–Vis spectrum of the ligand 1 displayed
a peak at 347 nm which can be related to the absorption
of energy for the intra-ligand n-π* transition. The com-
plexes exhibited charge transfer shoulder bands in the
range of 448–461 nm. A low energy band corresponding
to the forbidden d-d transition was observed in the range
of 612–624 nm for both vanadium and iron complexes.
The intra ligand peaks were retained with slight shifts in
the complexes.
FIGURE 3 Manually simulated EPR spectrum of the
vanadium (IV) species (2) with the characteristic hyperfine splitting
of 8 lines

ANTIDIABETIC AND BIOLOGICAL MEDIA STUDY OF V(IV) AND FE(III) COMPLEXES
7 of 12
TABLE 1 Spin-Hamiltonian parameters acquired from the EPR spectrum of [VIVO{(hpdbal)2-an}] (2) in DMSO
Ax = Ay = A⊥
Az = A
Equatorial
donor set
k
Sample
2
g
x=gy=g⊥
gz = g
giso
(10−4 cm−1
)
(10−4 cm−1
)
Aiso
Δg /Δg⊥
k
k
1.985
1.951
1.974
59.9
157.7
92.5
2.96
Namine, Namine,
OArO−, OArO
−
amine part of the ligand system between 190-320 ꢀC
followed by the loss of wꢀhole of organic ligand backbone
left in between 320-700 C. The residual corresponds to
the vanadium oxide material which is the end product of
the thermal decomposition of compound 2. The TGA
analysis of the binuclear sulphate bridged iron complex 3
(SI Figure 2) showed weight loss characteristic to the
predicted structure with the stepwise loss of coordinated
water molecules, amine part of the ligand and the whole
of ligand backbone in successions with the liberation of
sulphur dioxide gas (Scheme 5).
keeping the ligand {H2(hpdbal)2-an} (1) and standard
antidiabetic drug metformin as references. HepG2 cells
which were induced with insulin resistance have been
used as diabetic model for the study. With the addition of
insulin and 2-NBDG, the fluorescence reading given by
the cells added with different concentrations of the com-
pounds were noted. The readings were compared with
the fluorescence readings from control, test, standard,
reference and NBDG molecules alone.
The insulin resistant HepG2 cells showed low uptake
of NBDG thereby simulating diabetic environment.
When these cells were added with the ligand and com-
plexes, the NBDG uptake was higher. Notably, when the
vanadium complex, [VIVO{(hpdbal)2-an}] (2) of 0.1 μM
and 0.2 μM concentrations was added, it induced excel-
lent NBDG uptake of 80.8% and 95.4% respectively by the
insulin resistant cells. This uptake is quantified by the
histogram plot given in Figure 4. The increased glucose
3.2 | Glucose uptake activity studies
The vanadium complex [VIVO{(hpdbal)2-an}] (2) and iron
complex [{FeIII (OH2)((hpdbal)2-an)}2 μ2-SO4] (3) were
evaluated for their glucose intake inducing abilities by
SCHEME 5 Thermal decomposition pathway of the iron (III) species (3) as predicted by the TG analysis

8 of 12
KONGOT ET AL.
FIGURE 4 Histogram representing the
relative fluorescence readings which quantifies
the glucose uptake potentials. Here, NBDG is a
fluorescent D-glucose analogue used as a
tracker, Control is untreated human liver cancer
cells (HepG2), Test is the insulin resistant
HepG2 cells, Metformin is the standard
antidiabetic drug used as reference and 1, 2 and
3 are the compounds {H2(hpdbal)2-an},
[VIVO{(hpdbal)2-an}] and [{FeIII (OH2)
((hpdbal)2-an)}2 μ2-SO4] respectively
uptake potential induced by the V (IV) complex was
found to be higher than that induced by the standard
antidiabetic drug metformin (85.7% of NBDG uptake at
0.2 μM concentration). Although the Fe (III) complex did
not exhibit comparable activity as that of the vanadium
complex and standard, it displayed moderate activity
which was almost same as that shown by the ligand and
standard drug.
both the complexes (pKa for 2: 4.98; pKa for 3: 4.38) were
found to be in the most suitable range of any drug
candidate.[39]
From the UV–Vis spectrophotometric titration stud-
ies, it was found that the absorption maxima exhibited by
the compounds did not shift with varying pH conditions
(Figure 5 and SI Figure 4). Absorbance intensity shifts
were observed owing to protonation and deprotonation
effects on the compounds. The convenient pKa values
and the stability over wide pH range elucidates that both
the complexes (2 and 3) are bound to be stable in physio-
logical pH conditions prevalent in human serum.
3.3 | Biological media simulation studies
To supplement the bioactivity exhibited by the com-
plexes, their behavior in simulated biological media was
studied. Firstly, the effect of varied pH conditions (acidic
to basic) on the compounds were evaluated with the help
of potentiometric pH titrations and UV–Vis spectrophoto-
metric pH titrations. For this study, solutions of com-
plexes 2 and 3 were prepared in water-DMSO mixture
(50% (v/v)). The initial pH of the solution of compounds
was slowly varied in acidic and basic pH ranges of 3–9.
From the potentiometric titration readings, the
pharmaco-parameter pKa was calculated using the plot
of pH vs volume of base added (SI Figure 3). The pKa of
The behavior and interaction modes of the most
potent vanadium complex 2 with a model drug carrier
protein, BSA were evaluated with the support of emis-
sion, absorbance and IR spectra. For the emission and
absorbance studies, the spectra were measured for titra-
tions of ten different concentrations of vanadium com-
plex (0 μM to 10 μM) with a fixed concentration of BSA
solution (5 μM). The emission spectra were studied using
three techniques, viz., steady state, synchronous and
three dimensional. In the steady state fluorescence spec-
tra, it was observed that the emission profile of protein at
347 nm due to the amino acid residues got gradually
FIGURE 5 UV–Visible
spectrophotometric titration studies for
[VIVO{(hpdbal)2-an}] (2), the most active
antidiabetic compound in (a) acidic pH
and (b) basic pH

ANTIDIABETIC AND BIOLOGICAL MEDIA STUDY OF V(IV) AND FE(III) COMPLEXES
9 of 12
quenched with the addition of the compound (SI Figure
5). This quenching pattern was carefully evaluated with
the help of Stern-Volmer plot of F0/F vs C where F0 and
F are the emission intensities of pure BSA and BSA
titrated with compound 2 at 347 nm. The graph was
observed to be almost linear upon fitting with R2 value of
0.98 (Figure 6a). This gave a clue on static quenching of
the BSA fluorophore. The number of active binding sites
(n) for the compound to bind with BSA was calculated to
be 1.5 using a double log graph (SI Figure 6). For the syn-
chronous fluorescence study, wavelength difference (Δλ)
was set at 15 nm and 30 nm for recording the respective
fluorescence of tyrosine and tryptophan residues in BSA.
It was observed that, with the addition of vanadium com-
plex, the tryptophan emission intensity got quenched to a
higher extent than that of the tyrosine residue
(Figure 6b).
For the three dimensional fluorescence study, three
concentrations (1 μM, 5 μM and 10 μM) of the vanadium
complex were titrated with BSA (5 μM) by setting excita-
tion wavelength between 200 nm to 360 nm and emission
wavelength between 200 nm to 650 nm. A graph with 3
representative axes viz. emission and excitation intensi-
ties vs wavelength was obtained from each titration study
(Figure 7). These plots gave an evidence of slow
unfolding of the coiled secondary structure of protein. In
the Figure 7, peak 2 which arises due to the n ! π* tran-
sitions occurring in the protein backbone was found to
be quenched by a higher rate than that of the peak 1
which is due to the amino acid residue tryptophan. This
observation hence gives a conclusion that the protein sec-
ondary structure is prone to be affected with increasing
concentrations of the complex, especially above 1:1 molar
ratio of complex and protein. For better understanding of
this quenching, the quenching percentage after each
addition is summarized in a table in the supporting infor-
mation (SI Table 1).
binding between BSA and the bioactive vanadium com-
pound. At all temperatures, it was observed that the
absorbance maxima at 280 nm showed a trend of incre-
ment with the addition of increasing concentrations of
ꢀ
the complex. The representative spectra at 25 C is given
in Figure 8a. From this study, a double reciprocal plot of
A0/(A-A0) vs 1/c was drawn at all temperatures (Fig-
ure 8b). The values of binding constants (Ka) obtained
were high which concluded a strong binding. In addition,
the Ka value was found to decrease with temperature
which gives further proof for static quenching of BSA
fluorophore. The binding thermodynamic parameters,
enthalpy change (ΔH), entropy change (ΔS) and Gibbs'
free energy change (ΔG) were calculated from van't Hoff
plot (lnKa vs 1/T) and Gibbs' free energy relation
(ΔG = ΔH-TΔS). All these parameters were calculated to
be of negative values suggesting a spontaneous and exo-
thermic binding reaction occurring between BSA and the
compound and that this binding is mostly made of hydro-
gen bonded and van der Waals interactions.[40]
The absorbance exhibited by the vanadium complex
was overlapped with the emission spectrum of pure BSA
to get a plot shown in SI Figure 7. From this plot, energy
parameters such as efficiency of energy transfer (E), criti-
cal distance of proximity needed for efficient binding (R0)
and the vicinity (r) between quencher compound and
BSA fluorophore were calculated with the help of FRET
equations. All the values obtained were in correlation
with the FRET theory associated with non-radiative
energy transfer between excited state molecules of pro-
tein and ground state quencher molecules.
The vibrational spectra of BSA were studied with the
addition of vanadium compound to understand the
changes occurring to the amide I band pertaining to the
peptide backbone (Figure 9). The amide I band, when
deconvoluted, can be seen as a mixture of different sec-
ondary structures of the protein with the alpha-helical
form being very prominent. With the addition of com-
pound 1, upon deconvolution of the amide I band region,
it was observed that the alpha-helical form was slowly
The absorbance spectra were studied at fouꢀr different
ꢀ
ꢀ
ꢀ
temperatures, viz. 20 C, 25 C, 30 C and 35 C mainly
for interpreting the thermodynamics and extent of
FIGURE 6 Graphs obtained from
the emission spectra of BSA titrated
with successive concentrations of
oxovanadium salan complex. (a) Stern-
Volmer plot drawn using the steady
state fluorescence emission spectra and
(b) Plot drawn using the synchronous
fluorescence spectra measured for
tyrosine and tryptophan residues

10 of 12
KONGOT ET AL.
FIGURE 7 Three dimensional fluorescence quenching profile of (a) BSA with the addition of increasing concentrations of vanadium
complex (b, c, d). Peak 1 represents the fluorescence due to the amino acid tryptophan (π ! π* transitions) and peak 2 represents the
fluorescence due to transitions in the protein polypeptide backbone
FIGURE 8 (a) UV–Vis
spectrophotometric spectra of BSA
added with different concentrations of
vanadium complex. (b) Double
reciprocal plot for the calculation of
binding constant
converting into other secondary forms such as the β-
sheets thereby losing its coiled secondary structure and
causing denaturation. Although, it is to be noted that this
conversion was observed to be prominent at high concen-
trations of the complex (1:2 ratio of BSA and compound).
The study of interaction of vanadium complex with
BSA summarized that the potent drug candidate shows
extensive binding and quenching of BSA fluorophore and
does not denature the protein structure which thereby
concludes that it can bind efficaciously with drug carrier
protein.
This study also allows us to predict that the mode of
antidiabetic action of the oxovanadium (IV) complex is
most likely to be same as that exhibited by most of the
potent oxovanadium antidiabetic drug candidates previ-
ously developed.[41]The complex is expected to undergo
speciation in the first hand in biological media and bind
to serum carrier protein such as human serum transfer-
rin. This will then make it way through the biological
membrane to bind with the cysteine residue of protein
tyrosine phosphatase enzyme, the free end of which is
responsible for de-phosphorylation of the insulin receptor

ANTIDIABETIC AND BIOLOGICAL MEDIA STUDY OF V(IV) AND FE(III) COMPLEXES
11 of 12
FIGURE 9 Deconvoluted
amide I band region of (a) BSA
(100 μM); (b) BSA (100 μM) added
with [VIVO{(hpdbal)2-an}] (50 μM)
and (c) BSA (100 μM) added with
[VIVO{(hpdbal)2-an}] (100 μM)
subunit and blocking the signaling cascade for the uptake
of glucose by the glucose transporter in a type II diabetic
patient.
of many more potent antidiabetic drug candidates for safe
and effectual usage.
ACKNOWLEDGMENTS
Authors are grateful to Science and Engineering
Research Board (SERB), India [SB/FT/CS-100/2013] and
Department of Science and Technology (DST), India [BT/
PR10353/PFN/20/889/2013] for providing the necessary
financial help. Authors are also thankful to Nanomission
project [SR/NM/NS-20/2014] for providing with EDS
characterization facility and Centre for Nano and Mate-
rial Sciences, Jain University, India for providing with
the required infrastructure.
4 | CONCLUSIONS
In this study, novel vanadium (IV) and iron (III) com-
plexes tethered with a O2N2 salan ligand were developed
and
studied
for
their
antidiabetic
potential.
[VIVO{(hpdbal)2-an}] was found to excellently induce
fluorescent glucose uptake of 95.4% by the insulin resis-
tant cells whose activity was very much higher than the
standard drug metformin which is the most commonly
prescribed type II antidiabetic drug. [{FeIII (OH2)
((hpdbal)2-an)}2 μ2-SO4] exhibited moderate activity
which is almost similar to the metformin activity. Both
the compounds showed no significant changes in varying
pH media and exhibited optimum pKa values which sug-
gest their stability in biological pH conditions. The active
vanadium complex was found to interact with a carrier
protein BSA in an extensive manner through strong
interactions which further suggest that the potent drug
candidate can be easily transported in serum media
through the biological membrane barrier.
CONFLICT OF INTEREST
There are no conflicts to declare.
ORCID
Amit Kumar https://orcid.org/0000-0001-9760-6233
REFERENCES
[1] (a)Global report on diabetes, World Health Organization,
Geneva 2016. (b)IDF Diabetes Atlas 8th Edition 2017 Global
fact sheet, International Diabetes Federation, Belgium 2017.
[2] A. A. Tahrani, A. H. Barnett, C. J. Bailey, Nat. Rev. Endocrinol.
2016, 12, 566.
As an overall, the authors believe that this work will,
in future, pave the road for the design and development
[3] A. J. Krentz, C. J. Bailey, Drugs 2005, 65, 385.

12 of 12
KONGOT ET AL.
[4] T. Dujic, K. Zhou, L. A. Donnelly, R. Tavendale, C. N. Palmer,
E. R. Pearson, Diabetes 2015, 64, 1786.
[5] C. J. Bailey, Clin. Pharmacol. Ther. 2015, 98, 170.
[6] (a)E. O. Billington, A. Grey, M. J. Bolland, Diabetologia 2015,
58, 2238. (b)A. A. Tahrani, A. H. Barnett, C. J. Bailey, Lancet
Diabetes Endocrinol. 2013, 1, 140.
[32] R. A. Row, M. M. Jones, Inorg. Synth. 1957, 5, 113.
[33] (a)M. Kongot, N. Dohare, V. Singh, D. Reddy, N. K. Singhal,
R. Patel, A. Kumar, Eur. J. Pharm. Sci. 2018, 123, 335. (b)
M. Kongot, N. Dohare, D. S. Reddy, N. Pereira, R. Patel,
M. Subramanian, A. Kumar, J. Trace, Elem. Med. Biol. 2019,
51, 176.
[7] E. Ferrannini, R. A. DeFronzo, Eur. Heart J. 2015, 36, 2288.
[8] (a)M. Kongot, A. Kumar, Sci. Rep. 2018, 55, 41. (b)
T. Mukherjee, J. C. Pessoa, A. Kumar, A. R. Sarkar, Dalton
Trans. 2013, 42, 2594. (c)M. R. Maurya, C. Haldar, A. A. Khan,
A. Azam, A. Salahuddin, A. Kumar, J. C. Pessoa, Eur. J. Inorg.
Chem. 2012, 2012, 2560. (d)T. Mukherjee, J. C. Pessoa,
A. Kumar, A. R. Sarkar, Dalton Trans. 2012, 41, 5260.
[9] J. L. Domingo, M. Gomez, Food Chem. Toxicol. 2016, 95, 137.
[10] D. Rehder, Inorg. Chem. Commun. 2003, 6, 604.
[11] S. W. Taylor, B. Kammerer, E. Bayer, Chem. Rev. 1997, 97, 333.
[12] R. E. Berry, E. M. Armstrong, R. L. Beddoes, D. Collison, S.
N. Ertok, M. Helliwell, C. D. Garner, Angew. Chem. Int. Ed.
1999, 38, 795.
[34] C. Zou, Y. Wang, Z. Shen, J. Biochem. Biophys. Methods 2005,
64, 207.
[35] (a) N. H. Holyk, An electron paramagnetic resonance study of
model oxovanadium (IV) complexes in aqueous solution; Corre-
lation of magnetic properties with ligand type and metal struc-
ture, MS Thesis submitted to University of New Hampshire,
Durham, New Hampshire, 1979. (b)N. D. Chasteen, Biological
Magnetic Resonance, Plenum Press, New York 1981.
[36] G. R. Hausen, T. A. Kabanos, A. D. Keramidas, D. Mentzafos,
A. Terzis, Inorg. Chem. 1992, 31, 2587.
[37] E. Garribba, G. Micera, A. Panzanelli, D. Sanna, Inorg. Chem.
2003, 42, 3981.
[38] (a)M. Kongot, D. Reddy, V. Singh, R. Patel, N. K. Singhal,
A. Kumar, New J. Chem. 2019, 43, 10932. (b)M. Joseph,
A. Sreekanth, V. Suni, M. R. P. Kurup, Spectrochim. Acta A
Mol. Biomol. Spectrosc. 2006, 64, 637.
[13] (a)J. M. Winter, B. S. Moore, J. Biol. Chem. 2009, 284, 18577.
(b)M. R. Maurya, A. Kumar, J. C. Pessoa, Coord. Chem. Rev.
2011, 255, 2315.
[14] B. Lyonnet, X. Martz, E. Martin, Presse Med. 1899, 32, 191.
[15] C. E. Heyliger, A. G. Tahiliani, J. H. McNeill, Science 1985,
227, 1474.
[39] (a)H. Fischer, R. Gottschlich, A. Seelig, J. Membr. Biol. 1998,
165, 201. (b)D. T. Manallack, Perspectives in Med. Chem. 2007,
1, 25.
[16] J. H. McNeill, V. G. Yuen, H. R. Hoveyda, C. Orvig, J. Med.
Chem. 1992, 35, 1489.
[40] (a)B. Ojha, G. Das, J. Phys. Chem. B 2010, 114, 3979. (b)
M. Kongot, D. Reddy, V. Singh, R. Patel, N. K. Singhal,
A. Kumar, Spectrochim. Acta A Mol. Biomol. Spectrosc. 2019,
212, 330. (c)M. Kongot, N. Maurya, N. Dohare, M. Parray,
J. Kumar, A. Kumar, R. Patel, J. Biomol. Struct. Dyn. 2018, 36,
1161.
[41] (a)D. Rehder, Dalton Trans. 2013, 42, 11749. (b)E. Kioseoglou,
S. Petanidis, C. Gabriel, A. Salifoglou, Coord. Chem. Rev. 2015,
301, 87.
[17] K. H. Thompson, C. Orvig, Met. Ions Biol. Syst. 2004, 41, 221.
[18] K. H. Thompson, J. Lichter, C. LeBel, M. C. Scaife, J.
H. McNeill, C. Orvig, J. Inorg. Biochem. 2009, 103, 554.
[19] D. C. Crans, J. Inorg. Biochem. 2000, 80, 123.
[20] H. Sakurai, H. Sano, T. Takino, H. Yasui, J. Inorg. Biochem.
2000, 80, 99.
[21] B. A. Reul, S. S. Amin, J.-P. Buchet, L. N. Ongemba, D.
C. Crans, S. M. Brichard, Br. J. Pharmacol. 1999, 126, 467.
[22] N. Durai, G. Saminathan, J. Clin. Biochem. Nutr. 1997, 22, 31.
[23] J. W. Morgan, E. Anders, Proc. Natl. Acad. Sci. 1980, 77, 6973.
[24] A. C. Dlouhy, C. E. Outten, Met. Ions Life Sci. 2013, 12, 241.
[25] S. J. Lippard, J. M. Berg, Principles of Bioinorganic Chemistry,
University Science Books, Mill Valley 1994.
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
[26] K. P. Maier, H. Kopf, E. W. Neuse, J. Cancer Res. Clin. Oncol.
1984, 108, 336.
[27] M. Patra, G. Gasser, Nat. Rev. Chem. 2017, 1, 1.
[28] W. A. Wani, U. Baig, S. Shreaz, R. A. Sheikh, P. F. Iqbal,
E. Jameel, A. Ahmad, S. H. Mohd-Setapar, L. T. Hun, New J.
Chem. 2015, 40, 1063.
[29] R. K. Dubey, U. K. Dubey, S. K. Mishra, J. Coord. Chem. 2011,
64, 2292.
[30] R. Gust, D. Posselt, Metal salophen complexes for prevention
and treatment of cancer, WO 2008/090081, 2008.
[31] S. S. Mandal, K. I. Ansari, J. D. Grant, Apoptotic and anti-
tumor activities of metallosalens, Us 8198322, 2009.
How to cite this article: Kongot M, Reddy DS,
Singh V, Patel R, Singhal NK, Kumar A.
Oxidovanadium (IV) and iron (III) complexes with
O2N2 donor linkage as plausible antidiabetic
candidates: Synthesis, structural characterizations,
glucose uptake and model biological media studies.
Appl Organometal Chem. 2019;e5327. https://doi.
org/10.1002/aoc.5327